The kinetic parameters for penicillin G hydrolysis in systems with penicillin G acylase from Escherichia coli (free and immobilized on activated chitosan microbeads produced by electrostatic extrusion) were determined. The obtained kinetic results indicated that both systems (free and immobilized) are inhibited by high concentrations of the substrate (penicillin G) as well as by products of the reaction (6-aminopenicillanic acid and phenylacetic acid). The microbeads appeared convenient for penicillin G acylase immobilization reducing negative inhibitory effects. The hydrolysis was also investigated in a packed bed reactor. The derived kinetic model predicted good hydrolysis rates in the reactor while the system with recirculation of the reaction mixture proved to be a potentially favorable solution providing operation at low shear stresses and possibly higher hydrolysis rates than in the packed bed reactor alone.